We have shown that transplantation of undifferentiated embryonic stem (ES) cells results in improved left ventricular (LV) function after myocardial infarction (MI) in mice despite the fact that 80-90% of transplanted cells die and there is limited engraftment and differentiation. In vitro preliminary data indicate that factors released from ES cells are anti-apoptotic and anti-fibrotic. In vivo preliminary data suggest that transplantation of ES cells post-MI in C57Bl/6 mice results in cardiac regeneration as well as improved remodeling (including reduced apoptosis and fibrosis). Accordingly, we propose the overall hypothesis that these factors released from dying and surviving stem cells favorably influence myocardial remodeling and, ultimately, function. Our preliminary data indicate that TIMP-1 is anti-apoptotic in an in vitro model simulating the post-MI heart. Therefore, we also hypothesize that the favorable effects on remodeling of factors released from ES cells are mediated by both classic cell survival as well as TIMP-1 pathways. The specific questions we will address in this application are: 1) What factors are released from ES cells in cell culture and in vivo following post-MI transplantation and do they influence remodeling? 2) Is ES cell transplantation required to favorably influence remodeling or could ES cell-conditioned medium (CM) alone produce the same results? 3) What mechanisms are responsible for inhibition of apoptosis and fibrosis observed following CM or cell transplantation? Specific Aims are as follows; 1: Determine if ES-CM a) contains concentrations of released factors that inhibit apoptosis, and b) inhibits apoptosis via activation of phosphatidylinositol-3-OH kinase (PI3K/Akt1) and the extra-cellular signal regulated kinases (ERK1/2) survival pathways in vitro in the setting of oxidative stress simulating the post-MI heart. 2: Determine factors released from transplanted ES cells that attenuate remodeling post-MI. 3: Determine if intramyocardial injection of ES-CM attenuates remodeling post-MI. 4: Determine the mechanisms of inhibition of apoptosis and fibrosis following ES cell transplantation or ES-CM injection post-MI. Results of these studies will define the effect of transplanted ES cells or ES-CM on cardiac regeneration and remodeling and mechanisms of inhibition of apoptosis and fibrosis that may play a role in improved cardiac function. Identification and delineation of favorable effects of factors released from ES cells might lead to a new therapeutic strategy that could be an alternative to or combined with cell transplantation. Project Narrative The most common cause of heart disease in the U.S. is coronary artery disease and associated myocardial infarction, which results in the irreversible loss of functional heart muscle. The pharmacological agents (drugs) are unable to completely cure the disease. The results from the proposed projects will have long-term implications for future cell- based therapy to treat heart disease (s) using embryonic stem cells or potentially other donor cells.